The anti-platelet drug cilostazol enhances heart rate and interrenal steroidogenesis and exerts a scant effect on innate immune responses in zebrafish

Rationale Cilostazol, an anti-platelet phosphodiesterase-3 inhibitor used for the treatment of intermittent claudication, is known for its pleiotropic effects on platelets, endothelial cells and smooth muscle cells. However, how cilostazol impacts the endocrine system and the injury-induced inflammatory processes remains unclear. Methods We used the zebrafish, a simple transparent model that demonstrates rapid development and a strong regenerative ability, to test whether cilostazol influences heart rate, steroidogenesis, and the temporal and dosage effects of cilostazol on innate immune cells during tissue damage and repair. Results While dosages of cilostazol from 10 to 100 μM did not induce any noticeable morphological abnormality in the embryonic and larval zebrafish, the heart rate was increased as measured by ImageJ TSA method. Moreover, adrenal/interrenal steroidogenesis in larval zebrafish, analyzed by whole-mount 3β-Hsd enzymatic activity and cortisol ELISA assays, was significantly enhanced. During embryonic fin amputation and regeneration, cilostazol treatments led to a subtle yet significant effect on reducing the aggregation of Mpx-expressing neutrophil at the lesion site, but did not affect the immediate injury-induced recruitment and retention of Mpeg1-expressing macrophages. Conclusions Our results indicate that cilostazol has a significant effect on the heart rate and the growth as well as endocrine function of steroidogenic tissue; with a limited effect on the migration of innate immune cells during tissue damage and repair.


• Major Issues • Minor Issues
o Line 68: "…the stress response hormone catecholamine".Catecholamines are a class of molecule with an aromatic catechol and amine side-chain most notably including dopamine, epinephrine, and norepinephrine.Chromaffin cell granules contain mostly epinephrine and norepinephrine along with some dopamine (https://doi.org/10.1016/B978-0-12-416687-5.00030-0).Authors should revise their language to reflect the stress response hormones are epinephrine and norepinephrine, not "catecholamine".o Line 104 "It is generally accepted…" Please provide a citation.

• Major Issues
o Lines 135-6: Please verify that larval heartrate was recorded on high-speed video and analyzed offline in a slower playback.A human cannot accurately measure the reported heart rates between 90 and 230 bpm .Authors should also describe in detail how a single heartbeat was quantified.o Line 121 Animals: Please provide references to the golden and citrine strains utilized.o Lines 251-53: The authors are trying to compare their cortisol findings with their steroidgenic cell counts in an unclear way.They may wish to create a new last sentence stating something along the line of "increased cortisol levels are consistent with increased number of steroidgenic cells".

Discussion
• Major Issues • Minor Issues o Lines 347-8: "Cortisol is the most important endogenous glucocorticoid in both humans and teleosts".This is a completely subjective statement.o Line 351: Missing period.o Lines 398-400: The authors are slightly over stating their findings.Their study does not investigate the persistence of chronic inflammation into zebrafish adulthood, nor does it investigate changes in mental function of the fish.The authors should revise somewhat.Despite the images being duplicated, the red traced outline is slightly different between the two.The authors should explain and correct the error and ensure that duplicated data was not shared between the 50 and 100 μM dot plots.▪ 452: "50 and 100 μM cilostazol control groups".These are not control groups.

• Minor Issues
o Figure 5 is not mentioned explicitly in the text.

Figures•
Figures Lines 248 -250: Authors note that a low DMSO concentration increased measured cortisol levels.This accounts for approximately 30% of the claimed cortisol increase caused by 50 and 100 μM, a large amount.Additionally, the authors show that the DMSO control is significantly larger than the 30μM treatment group.The authors must address this difference.It is possible that inappropriate statistics are being used, or there is major batch variation causing false positive results.(Seefigure3comments).oLines281-2:See figure 4 comments.Duplicated data.oLines283-290:The authors have transformed their neutrophil stain data into a Lines 292-304: The authors should make specific mention to the actual datacontaining figure 5. o Lines 314-25: Why does DMSO vehicle significantly increase macrophagerecruitment over blank control?As with the neutrophil staining, the authors should consider a live real-time analysis of macrophage migration to discuss trends.•MinorIssueso Line 218: "…developing zebrafish may be similar to those on the mammalian adults."Strange phrasing.Rephrase to just say "similar in mammals".
• Minor Issues o Line 129: Authors should describe what percentage (maximal) of DMSO zebrafish are exposed to.High DMSO concentrations can affect heartrate.timeseries format.They mention a lower number of neutrophils present in all samples by 12hpa and another upregulation by 24 hpa.The authors use this data to claim cilostazol plays a role in neutrophil retention but not recruitment.However, previous studies using live tracking of neutrophil migration have not seen this resurgence phenomenon (PMID: 16926288) including a real-time live tracking study (PMID: 25763660).The authors should address this discrepancy.The authors should also consider performing a real-time live neutrophil tracking experiment in order to support their claim of cilostazol-mediated neutrophil retention.o o Lines 240-241: The authors desc ribe the cortisol extraction and quantification procedure in the methods, but they should also briefly mention the experiment again here before discussing results.